Electrode for active oxygen species and sensor using the electrode

ABSTRACT

An electrode for active oxygen species comprising a conductive component with a polymer membrane of a metal porphyrin complex formed on the surface is disclosed. The electrode for active oxygen species can detect active oxygen species such as superoxide anion radicals, hydrogen peroxide, and .OH and other active radical species (NO, ONOO—, etc.) in any environment including in vivo environment as well as in vitro environment. The electrode thus can be used for specifying various diseases and examining active oxygen species in food or in water such as tap water and sewage water.

TECHNICAL FIELD

The present invention relates to an electrode for active oxygen speciesin the living body such as superoxide anion radicals (O₂ ⁻.) and to asensor for measuring the concentration of active oxygen species usingthe electrode. More specifically, the present invention relates to anelectrode for active oxygen species and a sensor for measuring theconcentration of active oxygen species which can be applied to in vivomeasurement without using a large amount of enzymes and without causinga problem of enzyme deactivation.

BACKGROUND ART

Superoxide anion radicals (O₂ ⁻.), which are active oxygen species, areproduced in vivo by oxidation of xanthine, hypoxanthine, and the likeinto uric acid by xanthine•xanthine oxidase (XOD), reduction of oxygenby hemoglobin, and the like. The superoxide anion radicals have animportant role in the synthesis of physiologically active substances,bactericidal action, senility, and the like. Various active oxygenspecies derived from the superoxide anion radicals are reported to causevarious diseases such as cancer. Therefore, measuring the concentrationof the active oxygen species including superoxide anion radicals in theliving body is believed to be important for specifying these variousdiseases.

When there is no substrate, these superoxide anion radicals becomehydrogen peroxide (H₂O₂) and oxygen molecules (O₂) by a dismutationreaction as shown in the formula (1). This dismutation reaction consistsof formation of HO₂. by the addition of a proton to the superoxide anionradicals, formation of hydrogen peroxide and oxygen molecules by thereaction of HO₂ with oxygen molecules, and formation of hydrogenperoxide and oxygen molecules by collision of HO₂ radicals (formulas (1)-(4)).2H⁺+2O₂ ⁻.→H₂O₂+O₂   (1)H⁺+O₂ ⁻.→HO₂.   (2)HO₂.+O₂ ⁻.+H⁺→H₂O₂+O₂   (3)HO₂.+HO₂.→H₂O₂+O₂   (4)

In this reaction system, the superoxide anion radical functions as anelectron acceptor (oxidizing agent), an electron donator (reducingagent), and a hydrogen ion acceptor (base). The former two functionshave been applied to measuring the concentration of superoxide anionradicals. For example, the concentration of superoxide anion radicalswas measured using the reaction for converting ferricytochrome c(trivalent) into ferrocytochrome c (divalent), the reaction forproducing blue formazan from nitroblue tetrazolium (NBT), and thereaction for reducing tetranitromethane (TNM). All of these reactionswere carried out on an in vitro basis.

On the other hand, a method for quantitatively measuring theconcentration of superoxide anion radicals in vivo has beeninvestigated. For example, McNeil et al., Tariov et al., and Cooper etal. reported that the concentration of superoxide anion radicals can beelectrochemically determined by preparing an enzyme electrode (acytochrome c-immobilized electrode) by modifying the surface of a goldor platinum electrode with an enzyme, N-acetylcysteine, and immobilizinga protein such as cytochrome c, which is a metal protein having an ironcomplex referred to as hem as an oxidation-reduction center, via an S—Aubond (C. J. McNeil et al., Free Radical Res. Commun., 7, 89 (1989); M.J. Tariov et al., J. Am. Chem. Soc. 113, 1847 (1991); and J. M. Cooper,K. R. Greenough and C. J. McNeil, J. Electroanal. Chem., 347, 267(1993)).

The method is based on the following measurement principle. That is,cytochrome c (trivalent) (cyt.c(Fe³⁺)) reacts with superoxide anionradicals and is reduced to cytochrome c (divalent) (cyt.c(Fe²⁺))according to the reaction formula (5). Next, cytochrome c (divalent)reduced with O₂ ⁻ is electrochemically reoxidized according to thereaction formula (6). The oxidation current generated in this reactionis measured, whereby the concentration of the superoxide anion radicalsare quantitatively determined in an indirect manner.cyt.c(Fe³⁺)+O₂ ⁻→cyt.c(Fe²⁺)+O₂   (5)cyt.c(Fe²⁺)→cyt.c(Fe³⁺)+e-   (6)

However, since cytochrome c is an electron transfer protein which ispresent in vivo on intracellular mitochondrial membranes, a large numberof cells (e.g. 10⁵-10⁶ cells) is required to form an electrode on whichcytochrome c is immobilized in an amount sufficient for the measurement.In addition, the enzyme used is deactivated in several days. Therefore,development of an electrode that can detect active oxygen species suchas superoxide anion radicals without requiring a large amount of enzymesand without causing the problem of enzyme deactivation has been desired.

DISCLOSURE OF THE INVENTION

In view of this situation, the inventors of the present invention haveconducted extensive studies to obtain an electrode which can detectactive oxygen species such as superoxide anion radicals by anoxidation-reduction reaction. As a result, the inventors have found thatan electrode produced by forming a polymer membrane of a metal porphyrincomplex, formed by introducing a metal atom into the center of aporphyrin compound, on the surface of a conductive component does notrequire a large amount of enzymes, is free from the problem ofdeactivation, and can be applied to detecting active oxygen species andmeasuring their concentration.

Specifically, the present invention provides an electrode for activeoxygen species comprising a conductive component with a polymer membraneof a metal porphyrin complex formed on the surface.

The present invention further provides a sensor for measuring theconcentration of active oxygen species comprising an electrode foractive oxygen species comprising a conductive component with a polymermembrane of a metal porphyrin complex formed on the surface, a counterelectrode, and a reference electrode.

Furthermore, the present invention provides a method for detectingactive oxygen species in a sample comprising measuring the currentproduced between the metal in the metal porphyrin polymer membrane andthe active oxygen species using the above-described sensor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing an example of the three-electrode cell usedfor preparing the electrode of the present invention.

FIG. 2 is a drawing showing an example of the two-chamberthree-electrode cell used for preparing the electrode of the presentinvention.

FIG. 3 is a drawing showing an example of the needle-type electrode andthe two-chamber three-electrode cell used for preparing the electrode ofthe present invention, wherein (A) is the two-chamber three-electrodecell, (B) is the entire needle-type electrode, and (C) is the tip of theneedle-type electrode.

FIG. 4 is a drawing showing an improved needle-type electrode used forpreparing the electrode of the present invention. This electrode is animprovement of the needle-type electrode of FIG. 3, wherein (A) showsthe entire improved needle-type electrode and (B) is the tip of theimproved needle-type electrode.

FIG. 5 is a drawing showing an example of the measuring device used formeasuring active oxygen species.

FIG. 6 is a drawing showing an example of the measuring device used formeasuring active oxygen species.

FIG. 7 shows graphs of the UV-visible spectrum of H₂T3ThP (7(a)) andUV-visible spectrum of FeT3ThP (7(b)).

FIG. 8 shows graphs of the UV-visible spectrum of H₂T2AmP (8(a)) andUV-visible spectrum of FeT2AmP (8(b)).

FIG. 9 is a graph showing a CV curve during electrolytic polymerizationof FeT3ThP.

FIG. 10 is a graph showing a CV curve during electrolytic polymerizationin the preparation of the electrode of FeT2AmP.

FIG. 11 is a graph showing the change over time in the oxidation currentduring addition of XOD in Comparative Product 1.

FIG. 12 is a graph showing the change over time in the oxidation currentduring addition of XOD in Inventive Product 1.

FIG. 13 is a graph showing the relation between the (degree of XODactivity)^(1/2) and the amount of current increase in Inventive Product1 and Comparative Product 1.

FIG. 14 is a drawing showing the change over time in the current whenXOD in Inventive Product 4 was added to a concentration of 100 mU/ml.

FIG. 15 is a drawing showing the relation between the amount ofsuperoxide anion radicals and the amount of current change.

BEST MODE FOR CARRYING OUT THE INVENTION

The electrode for active oxygen species of the present invention(hereinafter referred to as “electrode”) comprises a conductivecomponent with a polymer membrane of a metal porphyrin complex formed onthe surface.

Any component commonly used for electrodes can be used as a conductivecomponent for the electrode of the present invention without specificlimitations. Examples include carbons such as glassy carbon (GC),graphite, pyrolytic graphite (PG), highly oriented pyrolytic graphite(HOPG), and activated carbon, noble metals such as platinum, gold, andsilver, and In₂O₃/SnO₂ (ITO). Of these, glassy carbon is particularlypreferable in view of economical efficiency, processability,lightweight, and the like. There are no specific limitations to theshape of the conductive component, inasmuch as such a shape is usable asan electrode. Various shapes such as a cylinder, square pillar, needle,and fiber can be used. A needle-like shape is preferable for measuringthe concentration of active oxygen species in vivo, for example.

A polymer membrane of a metal porphyrin complex is formed on the surfaceof the conductive component-in the present invention. As examples of themetal porphyrin complex used for producing the polymer membrane, thecompounds of the following formula (I) or (II) can be given.

wherein M is a metal selected from the group consisting of iron,manganese, cobalt, chromium, and iridium, at least one of the four Rs isa group selected from the group consisting of a thiofuryl group,pyrrolyl group, furyl group, mercaptophenyl group, aminophenyl group,and hydroxyphenyl group, and the other Rs represent any one of thesegroups, an alkyl group, an aryl group, or hydrogen.

wherein M and R are the same as defined above, at least one of the twoLs is a nitrogen-containing axial ligand such as imidazole and itsderivative, pyridine and its derivative, aniline and its derivative,histidine and its derivative, and trimethylamine and its derivative, asulfur-containing axial ligand such as thiophenol and its derivative,cysteine and its derivative, and methionine and its derivative, or anoxygen-containing axial ligand such as benzoic acid and its derivative,acetic acid and its derivative, phenol and its derivative, aliphaticalcohol and its derivative, and water, and the other L is any one ofthese axial ligands or a group without a ligand.

The metal porphyrin complex represented by the above formula (I) orformula (II) is a complex compound in which a metal atom is coordinatedto a porphyrin compound. This porphyrin compound is a cyclic compoundformed from four pyrrole rings of which the four methine groups arebonded together at the α-position and the four nitrogen atoms arepositioned face-to-face toward the center. A complex compound (a metalporphyrin complex) can be formed by inserting a metal atom into thecenter. To form this compound, a conventional method for producing ametal complex such as a method of introducing a metal atom into thecenter of porphyrin using metalation, for example, can be used. In thepresent invention, various metals such as iron, manganese, cobalt,chromium, and iridium can be used as the metal introduced into thecenter of the porphyrin compound.

A suitable metal atom can be selected according to the type of activeoxygen species to be measured. For example, iron, manganese, cobalt, andthe like are preferably used when superoxide anion radicals aremeasured; iron, cobalt, manganese, chromium, iridium, and the like arepreferably used when molecular oxygen is measured; iron, manganese, andthe like are preferably used when hydrogen peroxide is measured; andiron, manganese, and the like are preferably used when —OH, NO, ONOO⁻,and the like are measured.

The porphyrin compound used in the present invention is preferably aporphyrin compound of which at least one of the 5, 10, 15, and 20positions according to the position numbering of the IUPAC nomenclatureis substituted with a thiofuryl group, pyrrolyl group, furyl group,mercaptophenyl group, aminophenyl group, hydroxyphenyl group, or thelike, and the other positions are substituted with any one of thesegroups, an alkyl group, an aryl group, or hydrogen. The followingcompounds can be given as specific examples:

-   -   5,10,15,20-tetrakis(2-thiofuryl)porphyrin,    -   5,10,15,20-tetrakis(3-thiofuryl)porphyrin,    -   5,10,15,20-tetrakis(2-pyrrolyl)porphyrin,    -   5,10,15,20-tetrakis(3-pyrrolyl)porphyrin,    -   5,10,15,20-tetrakis(2-furyl)porphyrin,    -   5,10,15,20-tetrakis(3-furyl)porphyrin,    -   5,10,15,20-tetrakis(2-mercaptophenyl)porphyrin,    -   5,10,15,20-tetrakis(3-mercaptophenyl)porphyrin,    -   5,10,15,20-tetrakis(4-mercaptophenyl)porphyrin,    -   5,10,15,20-tetrakis(2-aminophenyl)porphyrin,    -   5,10,15,20-tetrakis(3-aminophenyl)porphyrin,    -   5,10,15,20-tetrakis(4-aminophenyl)porphyrin,    -   5,10,15,20-tetrakis(2-hydroxyphenyl)porphyrin,    -   5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin,    -   5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin,    -   [5,10,15-tris(2-thiofuryl)-20-mono(phenyl)]porphyrin,    -   [5,10,15-tris(3-thiofuryl)-20-mono(phenyl)]porphyrin, ]    -   [5,10-bis(2-thiofuryl)-15,20-di(phenyl)]porphyrin,    -   [5,10-bis(3-thiofuryl)-15,20-di(phenyl)]porphyrin,    -   [5,15-bis(2-thiofuryl)-10,20-di(phenyl)]porphyrin,    -   [5,15-bis(3-thiofuryl)-10,20-di(phenyl))porphyrin,    -   [5-mono(2-thiofuryl)-10,15,20-tri(phenyl)]porphyrin, and    -   [5-mono(3-thiofuryl)-10,15,20-tri(phenyl)]porphyrin.

Among the ligands represented by L in the compound of the formula (II),as examples of the imidazole derivative, methylimidazole,ethylimidazole, propylimidazole, dimethylimidazole, and benzimidazolecan be given; as examples of the pyridine derivative, methylpyridine,methyl pyridylacetate, nicotinamide, pyridazine, pyrimidine, pyrazine,and triazine can be given; as examples of the aniline derivative,aminophenol and diaminobenzene can be given; as examples of thehistidine derivative, histidine methyl ester, histamine, andhippuryl-histidyl-leucine can be given; as examples of thetrimethylamine derivative, triethylamine and tripropylamine can begiven; as examples of the thiophenol derivative, thiocresol,mercaptophenol, mercaptobenzoic acid, aminothiophenol, benzene dithiol,and methylbenzene dithiol can be given; as examples of the cysteinederivative, cysteine methyl ester and cysteine ethyl ester can be given;as examples of the methionine derivative, methionine methyl ester andmethionine ethyl ester can be given; as examples of the benzoic acidderivative, salicylic acid, phthalic acid, isophthalic acid, andterephthalic acid can be given; as examples of the acetic acidderivative, trifluoroacetic acid, mercaptoacetic acid, propionic acid,and butyric acid can be given; as examples of the phenol derivative,cresol and dihydroxybenzene can be given; and as examples of thealiphatic alcohol derivative, ethyl alcohol and propyl alcohol can begiven.

Various polymerization methods such as electrolytic polymerization,solution polymerization, and heterogeneous polymerization can be used inthe present invention to form a polymer membrane of a metal porphyrincomplex on the surface of the conductive component. Of these,electrolytic polymerization is preferable. Specifically, the polymermembrane of a metal porphyrin complex can be formed on the surface ofthe conductive component by polymerization. Polymerization is carriedout by two-electrode (working electrode and counter electrode)electrolysis or three-electrode (working electrode, counter electrode,and reference electrode) electrolysis, including three-electrodeconstant potential electrolysis, three-electrode constant currentelectrolysis, three-electrode reversible potential sweep electrolysis,and three-electrode pulse electrolysis, using a suitable supportingelectrolyte such as tetrabutylammonium perchlorate (TBAP: Bu₄NClO₄),tetrapropylammonium perchlorate (TPAP: Pr₄NClO₄), or tetraethylammoniumperchlorate (TEAP: Et₄NClO₄) in an organic solvent such asdichloromethane, chloroform, or carbon tetrachloride, using theconductive component as the working electrode, an insoluble electrodesuch as a noble-metal electrode (e.g. Pt electrode), a titaniumelectrode, a carbon electrode, or a stainless steel electrode as thecounter electrode, and a saturated calomel electrode (SCE), asilver-silver chloride electrode, or the like as the referenceelectrode.

The electrolytic polymerization is preferably carried out by reversiblepotential sweep electrolysis or the like using a three-electrode cell asshown in FIG. 1, for example. In FIG. 1, 1 indicates a cell container;2, a conductive component; 3, a counter electrode; 4, a referenceelectrode; 5, a metal porphyrin complex solution; 6, a potentiostat; and7, an X-Y recorder.

When using a high concentration metal porphyrin complex solution, atwo-chamber three-electrode cell as shown in FIG. 2, for example, may beused. In FIG. 2, numerals 1-7 indicate the same items as in FIG. 1, 8indicates an electrolyte solution, and 9 is a sample vial.

To produce a simplified electrode for active oxygen species (aneedle-type electrode), a needle-type electrode 10 and a three-electrodecell as shown in FIGS. 3(A) and 3(B), for example, may be used. In FIG.3, 1 indicates a cell container; 4, a reference electrode; 5, a metalporphyrin complex solution; 6, a potentiostat; 7, an X-Y recorder; 8, anelectrolyte solution; 9, a sample vial; 10, a needle-type electrode; 11,a counter electrode; 12, a tip of the conductive component (metalporphyrin polymer membrane area); 13, an electrical insulating material;and 14 a counter electrode wire.

As shown in FIG. 3(C), this electrode employs a counter electrode 11prepared by filling a small tube of an electrical insulating material 13with the conductive component and covering this small tube with themetal used as the counter electrode. The electrode can be used as aneedle-type electrode by forming a metal porphyrin polymer membrane onthe surface at the tip 12 of the conductive component.

The thickness of the polymer membrane of the metal porphyrin complex isappropriately determined according to the type of the electrode andmetal porphyrin complex and the type of active oxygen to be measured. Athickness of 1 μm or less is preferable from the viewpoint of electrodeactivity, modification stability, and the like.

To produce a simplified electrode for active oxygen species (an improvedneedle-type electrode), based on the simplified electrode for activeoxygen species (needle-type electrode) shown in FIG. 3, with anobjective of removing an unnecessary current in the living body, currentnoises, and the like and of improving sensitivity, signal/noise ratio(S/N ratio), and the like, an improved needle-type electrode 15 as shownin FIG. 4(A) and a three-electrode cell as shown in FIG. 3(A) may beused, for example. In FIG. 4, 11 indicates a counter electrode; 12, atip of the conductive component (metal porphyrin polymer membrane area);13, an electrical insulating material; 14, a counter electrode wire; 15,an improved needle-type electrode; 16, a ground; and 17, a ground wire.

As shown in FIG. 4(B), this electrode has a conductive componentinserted in an electrical insulating material 13 (two-layer structure).The electrical insulating material 13 is placed in a counter electrodematerial 11 (three-layer structure), the counter electrode material 11is housed in an electrical insulating material 13(four-layer structure),and finally, the outside of the resulting small tube is coated with amaterial such as a metal capable of functioning as a ground (five-layerstructure). The coating acts as the ground 16. The electrode can be usedas an improved simplified electrode for active oxygen species(improved/needle-type electrode) by forming a metal porphyrin polymermembrane on the surface at the tip 12 of the conductive component.

The thickness of the polymer membrane of the metal porphyrin complex isappropriately determined according to the type of the electrode andmetal porphyrin complex and the type of active oxygen to be measured. Athickness of 1 μm or less is preferable from the viewpoint of electrodeactivity, modification stability, and the like. Thisimproved/needle-type electrode can also be used for measuring compositematerials and the like. In such a case, it is possible to fabricate anelectrode having a structure of up to ten or more layers. As thematerial for the ground, a noble metal such as platinum, gold, titanium,stainless steel, and silver, a corrosion-resistant alloy such as aniron-chromium alloy, carbon, or the like can be used. Since the groundis frequently used in vivo, a material with a high safety such as anoble metal (e.g. platinum, gold, silver), titanium, stainless steel,and carbon is preferable.

To use the electrode of the present invention for measuring activeoxygen species, particularly for measuring the concentration of activeoxygen species, it is preferable to combine the electrode with (1) acounter electrode and a reference electrode (three-electrode type) or(2) a counter electrode (two-electrode type). As the material for thiscounter electrode, a noble metal such as platinum, gold, and silver,titanium, stainless steel, a corrosion-resistant alloy such as aniron-chromium alloy, carbon, or the like can be used. Since the counterelectrode is frequently used in vivo, a material with a high safety suchas a noble metal (e.g. platinum, gold, silver), titanium, and carbon ispreferable.

As the reference electrode, various reference electrodes such as asilver/silver chloride electrode and a mercury/mercuric chlorideelectrode can be usually used. A solid standard electrode can also beused.

A specific example of the measuring device that can be used formeasuring active oxygen species is shown in FIG. 5. In FIG. 5, numerals1, 3, 4, 6, and 7 indicate the same items as in FIG. 1, 18 indicates ameasuring electrode (working electrode), 19 indicates a microsyringe, 20indicates a solution to be measured, 21 indicates a magnetic stirrer,and 22 is a stirrer.

Another specific example of the measuring device used for measuringactive oxygen species is shown in FIG. 6. In FIG. 6, numerals 1, 6, 7,and 19-22 indicate the same items as in FIGS. 4 and 10 indicates aneedle-type electrode.

Although the electrode for active oxygen species of the presentinvention can be used as an electrode for detecting active oxygenspecies such as superoxide anion radicals using the above-describeddevice, the electrode can also be used as a sensor for measuring theconcentration of active oxygen species by using in combination with (1)a counter electrode and a reference electrode (three-electrode type) or(2) a counter electrode (two-electrode type). If the sensor formeasuring the concentration of active oxygen species of thisconfiguration is used in a system containing superoxide anion radicals,for example, the metal in the metal porphyrin complex forming thepolymer membrane is reduced by the superoxide anion radicals. Forexample, if the metal is iron, Fe³⁺ is reduced to Fe²⁺ by the superoxideanion radicals (formula (7)).

If the Fe²⁺ reduced by the superoxide anion radicals iselectrochemically reoxidized (formula (8)) while maintaining theelectrode for measuring the concentration at a potential (in the case ofthe three-electrode type (1)) or a voltage (in the case of thetwo-electrode type (2)) to a degree at which Fe²⁺ can be oxidized, thecurrent (oxidation current) flowing in this instance corresponds to theconcentration of the superoxide anion radicals. Therefore, theconcentration of the superoxide anion radicals dissolved in the samplesolution can be quantitatively detected from the oxidation current.Specifically, the concentration of the superoxide anion radicals can bedetermined based on the same principle of the above formulas (5) and(6). The quantitative detection based on this principle is also possiblefor active oxygen species such as hydrogen peroxide and -OH, otheractive radical species such as NO and ONOO—, and the like.Por(Fe³⁺)+O₂ ⁻.→Por(Fe²⁺)+O₂   (7)Por(Fe²⁺)→Por(Fe³⁺)+e-   (8)wherein “Por” indicates porphyrin.

Since the electrode for active oxygen species of the present inventionhas a polymer membrane of a metal porphyrin complex on the surface of aconductive component, the electrode is remarkably strong and free fromthe problem of deactivation as compared with a conventional cytochromec-immobilised electrode. In addition, since the polymer membrane ofmetal porphyrin is formed by electrolytic polymerization or the like,preparation of the electrode of the present invention is very easy ascompared with a conventional electrode. The electrode of the presentinvention can be produced in a shape particularly suitable forapplication in vivo, for example, a needle-like shape.

In this manner, the electrode for active oxygen species of the presentinvention can not only detect active oxygen species such as superoxideanion radicals, hydrogen peroxide, and .OH and other active radicalspecies (NO, ONOO—, etc.), but also quantitatively measure these activeoxygen species by combining with a counter electrode and referenceelectrode in any environment including in vivo environment as well as invitro environment. The electrode of the present invention therefore canbe used widely in various fields.

Specifically, since various diseases can be specified by active oxygenspecies and other active radical species in vivo, a disease such ascancer can be detected by, for example, measuring the concentration ofactive oxygen species in blood.

On the other hand, with regard to the application in in vitroenvironment, decomposition conditions of food can be observed bymeasuring active oxygen species and their concentration in food. Waterpollution conditions can also be observed by measuring active oxygenspecies and their concentration in tap water and sewage water.

Furthermore, the concentrations of superoxide anion radicals andsuperoxide dismutase (SOD), which is an enzyme with a function ofeliminating the anions, can be measured by determining the extinctiondegree of the superoxide anion radicals when a sample containing the SODis added.

EXAMPLES

The present invention will be described in more detail by referenceexamples, examples, and test examples, which should not be construed aslimiting the present invention.

Reference Example 1 Synthesis of5,10,15,20-tetrakis(3-thiophenyl)porphyrin (H₂T3ThP)

A 100 ml round bottom flask was charged with 50 ml of proprionic acid,2.0 ml of 3-thiophenecarbaldehyde, and 1.4 ml of pyrrole. The mixturewas refluxed for one hour at 160° C. while stirring. After refluxing,the reaction product was allowed to cool to room temperature, furthercooled with ice, and added to 200 ml of cold methanol. The mixture wasfiltered by suction. The filtrate was washed with methanol and purifiedusing silica gel chromatography (developing solvent: chloroform). Thesolvent was evaporated to dryness and the solid was recrystallized anddried under reduced pressure to obtain H₂T3ThP as black powder crystals(yield: 0.63 g, 19%). The product was identified using a UV-visiblespectrum photometer (UV-2100, manufactured by Shimadzu Corp.) and by¹H-NMR measurement. The results are shown in Tables 1 and 2.

Reference Example 2 Synthesis of5,10,15,20-tetrakis(2-aminophenyl)porphyrin (H₂T2AmP)

A 2 L four-necked flask equipped with a reflux condenser was chargedwith 500 ml of propionic acid. After addition of 25 g of2-nitrobenzaldehyde, the mixture was heated while refluxing at 110° C.with stirring. 12 ml of pyrrole was added and the mixture was refluxedat the boiling point for 30 minutes. After addition of 50 ml ofchloroform, the mixture was cooled with ice and filtered by suction. Thefiltrate was washed with 400 ml of chloroform and dried under reducedpressure (100° C., six hours, 0.1 kPa) to obtain a precursor5,10,15,20-tetrakis(2-nitrophenyl)porphyrin (H₂T2NO₂P) as black purplecrystals (yield: 5.0 g, 14%).

A 2 L four-necked flask was charged with 300 ml of 12 N HCl. Afteraddition of 5.0 g of 5,10,15,20-tetrakis(2-nitrophenyl)porphyrin(H₂T2NO₂P) synthesized as described above and 20.0 g of tin (II)chloride dihydrate, the mixture was heated at 65-70° C. for 30 minutes.Aqueous ammonia was gradually added and the mixture was filtered bysuction. The filtrate was dried under reduced pressure. A 2 L beaker wascharged with the filtrate. The filtrate was extracted with 10 L ofacetone and the extract was evaporated to dryness using an evaporator.The dry solid was dissolved in 2 L of chloroform. The solution waswashed with aqueous ammonia and ion exchange water and dehydrated withanhydrous sodium sulfate. The solvent was evaporated to dryness using anevaporator and the solid was recrystallized and dried under reducedpressure to obtain H₂T2AmP as purple crystals (yield: 4.0 g, 90%). Theproduct was identified using a UV-visible spectrum photometer (UV-2100,manufactured by Shimadzu Corp.) and by ¹H-NMR measurement in the samemanner as in Reference Example 1. The results are shown in Tables 1 and2. FIG. 7(a) shows the UV-visible spectrum of H₂T3ThP and FIG. 8(a)shows the UV-visible spectrum of H₂T2AmP.

(Identification Results) TABLE 1 Porphyrin δ _(H)(CDCl₃/TMS, ppm)

−2.7(s,2H,pyrrole-NH) 7.7(t,4H,thiophene-H) 8.0(m,8H,thiophene-H)8.9(s,8H ,pyrrole-β-H)

−2.7(s,2H,pyrrole-NH) 4.0(s,8H,amino-H) 7.0-7.7(m,16H,phenyl-H)8.9(s,8H,pyrrole-β-H)

(Identification Results) TABLE 2 λ_(max)(nm) Porphyrin Soret band Q bandH₂T3ThP 422 519 556 594 651 FeT3ThP 425 516 H₂T2AmP 420 516 549 590 652FeT2AmP 425 493

The results of identification for H₂T3ThP and H₂T2AmP in Table 1confirmed peaks of protons forming the porphyrin ring and peaks specificto the porphyrin compounds. The peak of H₂T2AmP was confirmed at 4.0ppm, which was assumed to be the peak of a proton forming an aminogroup. The results in Table 2 and FIGS. 7(a) and 8(a) confirmed theUV-visible spectra based on H₂T3ThP and H₂T2AmP. The above resultsconfirmed that H₂T3ThP and H₂T2AmP were synthesized in ReferenceExamples 1 and 2.

Reference Example 3 Synthesis of metal porphyrin complex (1)(introduction of central metal (Fe) into H₂T3ThP by metalation)

A 50 ml three-necked flask was charged with 10 ml of 48% hydrobromicacid. After injection of nitrogen gas for 30 minutes, 100 mg of reducediron was added. The mixture was stirred at 100° C. until the reducediron was dissolved. After the stirring, the solvent was evaporated underreduced pressure to obtain iron bromide anhydride as white powder.

250 mg of porphyrin (H₂T3ThP) prepared in Reference Example 1 and 200 mlof dimethylformamide (DMF) to which nitrogen gas was previously injectedfor 30 minutes were added to the product. The mixture was reacted in anitrogen atmosphere for four hours. After the reaction, 200 ml ofchloroform was added to the reaction product. The mixture was washedwith ion exchange water, dehydrated with anhydrous sodium sulfate, andfiltered. The solvent was evaporated using an evaporator and the solidobtained was purified using alumina column chromatography (developingsolvent:

-   -   chloroform/methanol=20/1). After adding 48% hydrobromic acid to        the eluate, the mixture was dehydrated with anhydrous sodium        sulfate and filtered. The solvent was evaporated and the solid        was recrystallized and dried under reduced pressure to obtain        metal porphyrin (H₂T3ThP containing Fe at the center;        hereinafter referred to as “FeT3ThP”) as black crystals (yield:        230 mg, 84%).

Reference Example 4 Synthesis of metal porphyrin complex (2)(introduction of central metal (Fe) into H₂T2AmP by metalation)

Black crystals of a metal porphyrin complex containing Fe at the centerof H₂T2AmP (hereinafter referred to as “FeT2AmP”) were obtained in thesame manner as in Reference Example 3, except for using 250 mg ofH₂T2AmP (obtained in Reference Example 2) as a porpherin compound(yield: 224 mg, 83%). The products of Reference Examples 3 and 4 wereidentified using a UV-visible spectrum photometer. The results are shownin FIGS. 7 and 8 and Table 2.

FIG. 7(a) shows the UV-visible spectrum of H₂T3ThP and FIG. 7(b) showsthe UV-visible spectrum of FeT3ThP. FIG. 8(a) shows the UV-visiblespectrum of H₂T2AmP and FIG. 8(b) shows the UV-visible spectrum ofFeT2AmP. Table 3 shows the results of measuring the UV-visible spectra.

The porphyrin compounds in which a metal is not coordinated at thecenter (H₂T3ThP and H₂T2AmP) have a peak based on the conjugate ring atnear 400 nm. The molecular extinction coefficient in the peak is3.6−6.0×10⁵ M⁻¹ cm⁻¹. The peak is called a Soret band.

In addition, the porphyrin compounds have four peaks called Q bands ofwhich the molecular extinction coefficient is 10^(4 M) ^(−1 cm) ⁻¹ inthe visible area. Introduction of a metal is confirmed generally byusing spectral changes of the Q bands. Comparison of (a) with (b) inFIG. 7 and Table 2 indicates that there are four peaks of Q bands in(a), whereas the number of peaks is reduced to one in (b). This is inagreement with a typical behavior when porphyrin forms a metal complex.Accordingly, it was confirmed that porphyrin formed a complex with iron,whereby a metal porphyrin complex was synthesized.

Reference Example 5 Synthesis of metal porphyrin complex (3)(introduction of central metal (Mn) into H₂T3ThP by metalation)

A 300 ml four-necked flask equipped with a reflux condenser with anargon balloon attached to the upper part, which can be heated over anoil bath and stirred using a magnetic stirrer, was charged with 100 mlof DMF containing 0.5 g of manganese acetate and 0.5 g of H₂T3ThPobtained in Reference Example 1. After saturation with argon, themixture was refluxed at 140° C. for one hour.

The resulting solution was poured into a separating funnel and washedwith chloroform and ion exchange water. After addition of anhydrousmagnesium sulfate, the mixture was dehydrated for one hour and filtered.The filtrate was evaporated to dryness using an evaporator.

The dry solid was separated and purified using column chromatography(filler: basic alumina, eluate: chloroform/methanol=20/1). After thefiltration, the filtrate was evaporated to dryness. The solid was driedunder reduced pressure (0.1 kPa, 100° C.) to obtain MnT3ThP as blackpurple crystals (yield: 0.36 g).

The UV-visible absorption spectrum was measured to confirm introductionof a metal. The absorption peaks of MnT3ThP were confirmed at 380 nm,405 nm, 480 nm, 533 nm, 583 nm, and 623 nm, which differed from theabove-described case of H₂T3ThP. Introduction of a metal was thusconfirmed.

Reference Example 6 Synthesis of metal porphyrin complex having axialligand (1) (synthesis of FeT3ThP to which 1-methylimidazole iscoordinated)

FeT3ThP obtained in Reference Example 3 (0.018 g) and 1-methylimidazole(2-100 μl) (FeT3ThP: 1-methylimidazole=1-50 (molar ratio)) were added to0.5 ml of dichloromethane. The mixture was stirred while irradiatingultra sonic wave (15 W) for five minutes or not irradiating ultrasonicwave for six hours or more.

The UV-visible absorption spectrum was measured to confirm coordinationof a ligand. An absorption peak of the complex obtained by coordinating1-methylimidazole to FeT3ThP was generated at 421 nm, which differedfrom the above-described case of FeT3ThP. Coordination of a ligand wasthus confirmed.

The complex was evaporated to dryness using an evaporator and stored, orused for preparing an electrode for active oxygen species.

Example 1 Preparation of Electrode for Active Oxygen Species (1)

A glassy carbon (GC) electrode (diameter: 1.0 mm, manufactured by BASInc.) was polished using an alumina polishing agent (0.05 μm). Afterwashing with water, the electrode was further washed with methanol. Apolymer membrane was formed on the surface of this electrode byelectrolytic polymerization using the following electrolytic solutionand procedure to prepare a glassy carbon electrode with a polymermembrane of FeT3ThP formed on the surface (Inventive Product 1) and aglassy carbon electrode with a polymer membrane of FeT2AmP formed on thesurface (Inventive Product 2).

(Sample Solution)

As a metal porphyrin complex, FeT3ThP or FeT2AmP synthesized inReference Example 3 or 4 in a solution with a concentration of 0.05 Mwas used. As a solvent, (anhydrous) dichloromethane containing 0.1 Mtetrabutylammonium perchlorate (Bu₄NClO₄/TBAP) as a supportingelectrolyte was used. Oxygen dissolved in the solvent was removed usingargon gas.

(Procedure)

Electrolytic polymerization was carried out by reversible potentialsweep electrolysis using a three-electrode cell having a configurationshown in FIG. 1 (working electrode: GC, counter electrode: Pt line,reference electrode: SCE). The sweep range was 0 to 2.0 V for SCE in thecase of preparing an electrode for active oxygen species using FeT3ThP;the range was −0.2 to 1.4 V for SCE in the case of preparing anelectrode for active oxygen species using FeT2AmP. The sweep rate was0.05 V/s in both cases. The number of times of sweep was once in thecase of FeT3ThP and three times in the case of FeT2AmP. The cyclicvoltammogram obtained in this electrolytic procedure (CV curve) wasrecorded in a X-Y recorder (manufactured by Riken Denshi Co., Ltd.). Theresults are shown in FIGS. 9 and 10.

FIG. 9 shows a CV curve during electrolytic polymerization whenpreparing an electrode using FeT3ThP as a metal porphyrin complex. Basedon this curve, it is assumed that cationic radicals of thiophene areproduced at +1.74 V (for SCE). Since almost no cathode current due toreduction of the cationic radicals flows when reversing the potentialsweep, it is assumed that the produced cationic radicals are immediatelypolymerized in the solution. The reduction peak at near 0.6 V (for SCE)is assumed to be a redox response of the polymer. The results confirmedthat a polymer membrane of the metal porphyrin complex (FeT3ThP) wasformed on the surface of the GC electrode.

FIG. 10 shows a CV curve during electrolytic polymerization whenpreparing an electrode using FeT2AmP as a metal porphyrin complex. Basedon this curve, it is assumed that cationic radicals of aniline(aminobenzene) are produced at +1.02 V (for SCE). Since almost nocathode current due to reduction of the cationic radicals flows whenreversing the potential sweep, it is assumed that the produced cationicradicals are immediately polymerized in the solution. The reduction peakat near 0.1 V (for SCE) is assumed to be a redox response of thepolymer. The same peak was found in the second sweep. Further progressof the polymerization reaction was thus confirmed. The reduction peak atnear 0.23 V (for SCE) is assumed to be a redox response of the polymer.The results confirmed that a polymer membrane of the metal porphyrincomplex (FeT2AmP) was formed on the surface of the GC electrode.

Example 2 Preparation of Electrode for Active Oxygen Species (2)

A glassy carbon (GC) electrode surrounded by polyether ether ketone(PEEK), with only one end being exposed (PEEK diameter: 3 mm, GCdiameter: 1 mm, end area: 0.0079 cm²; the other end made of brass) wasprovided. The GC end of the elecrtrode was polished sequentially by 6 μmpolishing diamond and 1 μm polishing diamond. After finish polishing onan alumina polishing pad using an alumina polishing agent (0.05 μm), theelectrode was washed with ion exchange water and acetone.

0.0037 g of FeT2AmP obtained in Reference Example 4 and 0.171 g of TBAPwere put into a 5 ml measuring flask. Acetonitrile was added to themixture to make the total volume 5 ml, thereby obtaining a samplesolution.

The sample solution was put into a cell vial. A three-electrodeelectrochemical cell with a GC electrode as a working electrode shown inFIG. 1 was fabricated. The atmosphere was replaced with argon.

The potential sweep was carried out three times in the potential sweeprange of −0.3 to 1.0 V for Ag/Ag⁺ at a potential sweep rate of 200mV/sec. The sweep initiation potential and the sweep terminationpotential were 0 V for Ag/Ag⁺. The sweep was carried out first in thenegative direction. After the sweep, the cell was washed sequentiallywith acetonitrile and ion exchange water to prepare a glassy carbonelectrode with a polymer membrane of FeT2AmP formed on the surface(Inventive Product 3).

Example 3 Preparation of Electrode for Active Oxygen Species (3)

0.171 g of TBAP was put into a 5 ml measuring flask. Dichloromethane wasadded to make the total volume 5 ml, thereby obtaining an electrolyticsolution.

0.0182 g of FeT2AmP obtained in Reference Example 4, 0.0171 g of TBAP,and 0.5 ml of dichloromethane were added to a sample vial, while the tipof the sample vial sealed with vycor glass was immersed in theelectrolytic solution.

A Pt counter electrode and the GC electrode used in Example 2 were putinto the sample vial and an Ag/Ag⁺ electrode was disposed on the outerside of the sample vial as shown in FIG. 2 to fabricate a two-chamberthree-electrode electrochemical cell. The atmosphere in the two chamberswas replaced with argon.

The potential sweep was carried out three times in the potential sweeprange of −0.3 to 2.5 V for Ag/Ag⁺ at a potential sweep rate of 50mV/sec. The sweep initiation potential was 0 V for Ag/Ag⁺ and the sweeptermination potential was −0.3 V for Ag/Ag⁺. The sweep was carried outfirst in the negative direction. After the sweep, the cell was washedsequentially with dichloromethane and ion exchange water to prepare aglassy carbon electrode with a polymer membrane of FeT2AmP formed on thesurface (Inventive Product 4).

Example 4 Preparation of Electrode for Active Oxygen Species (4)

An electrode rod of glassy carbon (diameter: 0.28-0.30 mm) wasintroduced into a glass capillary (internal diameter: 0.3 mm). Theseproducts were introduced into a needle (made of platinum, stainlesssteel, or the like corresponding to about 18 G injection needle). Theseproducts were joined and secured using an epoxy adhesive, acrylicadhesive, manicure, or the like. Next, the glassy carbon and the outsideneedle were respectively joined with a platinum electrode, stainlesssteel electrode, copper electrode, or the like via a conductive adhesivesuch as a silver paste or carbon paste. The tip was polished using agrinder to prepare a needle-type electrode with a three-layer structureas shown in FIG. 3 (glassy carbon: working electrode, inner glasscapillary: insulation part between working electrode and counterelectrode, outer needle: counter electrode).

An electrolytic solution (5 ml) prepared in the same manner as inExample 3 was put into a cell container. A dichloromethane solution ofFeT3ThP obtained in Reference Example 6 to which 1-methylimidazole wascoordinated containing 0.0171 g of TBAP was added to a sample vial,while the tip of the sample vial sealed with vycor glass was immersed inthe electrolytic solution.

The needle-type electrodes (working electrode and counter electrode)prepared as described above were put into the sample vial and an Ag/Ag⁺electrode was disposed on the outer side of the sample vial as shown inFIG. 3 to fabricate a two-chamber three-electrode electrochemical cell.

The cell was electrolytically polymerized using a reversible potentialsweep method (potential sweep range: −0.1 to +2.0 V for Ag/Ag⁺,potential sweep rate: 10 to 500 mV/sec) and a constant potential method(potential: +2.0 V for Ag/Ag⁺) for 5-120 minutes. After the electrolyticpolymerization, the cell was washed sequentially with dichloromethaneand ion exchange water to prepare a needle-type electrode with athree-layer structure including a glassy carbon electrode with a polymermembrane of FeT3ThP to which 1-methylimidazole was coordinated on thesurface (glassy carbon surface-modified by polymer membrane: workingelectrode, inner glass capillary: insulation part between workingelectrode and counter electrode, outer needle: counter electrode;Inventive Product 5).

Comparative Example 1 Preparation of Cytochrome c-immobilized GoldElectrode

As a comparative product for the electrodes of the inventive products, acytochrome c-immobilized gold electrode as an electrode for determiningthe concentration of superoxide anion radicals was prepared in thefollowing manner.

A gold electrode (diameter: 1.6 mm, manufactured by BAS Inc.) waspolished using an alumina polishing agent (0.05 μm) and washed withwater. The electrode was electrochemically treated in 1 M H₂SO₄ andwashed with water. Next, the electrode was immersed in 10 mM3-mercaptopropionic acid (hereinafter abbreviated to MPA, manufacturedby Aldrich Co.) (solvent: 10 mM phosphoric acid buffer solution (pH7.0)) for 24 hours to prepare a MPA-modified gold electrode. Afterwashing the electrode with water, the potential sweep was carried out in0.48 mM cytochrome c (type IV from Horse Heart, manufactured by SigmaCo.) (solvent: 10 mM phosphoric acid buffer solution (pH 7.0)) for 30minutes (sweep range: −0.4 to 0.4 V (for Ag/AgCl), sweep rate: 0.05V/s).

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, manufactured byPierce Chemical Co.) was added to make the final concentration 0.5 M andthe electrode was immersed in the mixture for two hours. The electrodewas finally washed with a phosphoric acid buffer solution to obtain acytochrome c-immobilized gold electrode (Comparative Product 1). Theabove phosphoric acid buffer solution was a mixture solution of disodiumhydrogen phosphate and sodium dihydrogen phosphate. Water distilled oncewas used for adjusting the sample solution.

Test Example 1

Measurement of Amount of Superoxide Anion Radicals (1)

The amount of superoxide anion radicals was measured using InventiveProduct 1 and Comparative Product 1.

First, the electrode of Inventive Product 1 or Comparative Product 1 asa working electrode and a Pt electrode as a counter electrode were putinto a cell container and a silver/silver chloride electrode (Ag/AgCl)was used as a reference electrode to form a three-electrode cell for thetest. An apparatus shown in FIG. 5 was prepared using thethree-electrode cell for the test at the center. In this test, thepotential sweep range was set at −0.2 to 0.25 V (for Ag/AgCl) for theelectrode of Comparative Product 1 and at −0.5 to 0.5 V (for Ag/AgCl)for the electrode of Inventive Product 1. Measurements were carried outusing several sweep rates.

A 2 mM aqueous potassium hydroxide solution containing 14.4 mM xanthine(manufactured by Sigma Co.) and a 10 mM Tris buffer solution containing10 mM potassium chloride (pH 7.5) were prepared. 0.365 ml of the formerand 14.635 ml of the latter were mixed to prepare a 0.35 mM xanthinesolution, which was used as a test solution. Oxygen dissolved in thetest solution was removed using high-purity argon gas.

Second, the test solution was added to the three-electrode cell for thetest. A potential of 0.2 V (for Ag/AgCl) which is sufficiently higherthan the oxidation-reduction potential of each electrode was applied.Xanthine oxidase (XOD, Grade III from butter milk, manufactured by SigmaCo.) was added to the test solution to make the final concentration0-100 mU/ml. The change over time in the oxidation current was recorded.The results are shown in FIG. 12 for Inventive Product 1 and FIG. 11 forComparative Product 1. XOD had been dialyzed with a 10 ml phosphoricacid buffer solution (pH 7.0) before use. All measurements were carriedout at room temperature.

XOD was added to xanthine to produce superoxide anion radicalsdose-dependently. FIG. 11 showing the change over time in the oxidationcurrent during addition of XOD in Comparative Product 1 as the controlindicates that the oxidation current rapidly increases immediately afterthe addition of XOD and is maintained almost at a constant value. FIG.12 showing the change over time in the oxidation current during additionof XOD in Inventive Product 1 indicates that the oxidation currentrapidly increases immediately after the addition of XOD as in FIG. 11and the current once decreases and is maintained almost at a constantvalue and that the current value depends on the concentration of XOD,specifically, the amount of superoxide anion radicals.

FIG. 13 is a graph showing the relation between the (degree of XODactivity)^(1/2) and the amount of current increase when XOD is added inInventive Product 1 and Comparative Product 1. The XOD activity wasdetermined taking into consideration the values in the documents ofFridovich et al. and Cooper et al. (J. M. McCord and I. Fridovich, J.Boil. chem., 243, 5753 (1968), J. M. McCord and I. Fridovich, J. Boil.chem., 244, 6049 (1969), I. Fridorich, J. Boil. chem., 245, 4053 (1970),and J. M. Cooper, K. R. Greenough, and C. J. McNeil, J. Electroanal.Chem., 347, 267 (1993)).

The results show that the amount of current increase in InventiveProduct 1 is proportional to the (degree of XOD activity)^(1/2) andthere is a linear relation between them in the same manner as inComparative Product 1 used as the control. These facts confirmed thatthe concentration of superoxide anion radicals can be measured using theelectrode for active oxygen species of the present invention.

Test Example 2 Measurement of Amount of Superoxide Anion Radicals (2)

The amount of superoxide anion radicals was measured using the electrodeof Inventive Product 5.

The electrode of Inventive Product 5, specifically, a needle-typeelectrode with a three-layer structure (glassy carbon surface-modifiedwith polymer membrane: working electrode, inner glass capillary:insulation part between working electrode and counter electrode, outerneedle: counter electrode; Inventive Product 5) was measured using atwo-electrode method. As a solution for measurement, a Tris buffersolution containing 0.15 mM xanthine (pH 7.5) was used. 0-100 mU/ml ofXOD was added to the solution. The applied voltage was 0-1.0 V.

The change over time in the current when XOD was added to aconcentration of 100 mU/ml at an applied voltage of +0.5 V is shown inFIG. 14. A graph of the relation between the amount of superoxide anionradicals and the amount of current change prepared by the peak currentvalue is shown in FIG. 15.

The correlation coefficient between the amount of superoxide anionradicals and the amount of current change determined from FIG. 15 was0.995. This indicates that the electrode of the present invention can beeffectively used for measuring the amount of superoxide anion radicals.

Industrial Applicability

The electrode for active oxygen species of the present invention candetect active oxygen species such as superoxide anion radicals, hydrogenperoxide, and -OH and other active radical species (NO, ONOO—, etc.) inany environment including in vivo environment as well as in vitroenvironment. In addition, it is possible to quantitatively determinethese active oxygen species and other active radical species bycombining the electrode with a counter electrode or a referenceelectrode. The electrode thus can be widely used in various fields.

For example, if used in vivo, various diseases can be specified fromactive oxygen species and other active radical species in the livingbody.

On the other hand, if used in vitro, active oxygen species and theirconcentration in food can be measured, based on which decompositionconditions of the food can be judged. Water pollution conditions canalso be observed by measuring active oxygen species and theirconcentration in tap water and sewage water.

1. (Deleted)
 2. An electrode for active oxygen species comprising aconductive component with a polymer membrane of a metal porphyrincomplex shown by the following formula (I) or (II) formed on thesurface,

wherein M is a metal selected from the group consisting of iron,manganese, cobalt, chromium, and iridium, at least one of the four Rs isa group selected from the group consisting of a thiofuryl group,pyrrolyl group, furyl group, mercaptophenyl group, and aminophenylgroup, and the other Rs represent any one of these groups, an alkylgroup, an aryl group, or hydrogen,

wherein M and R are the same as defined above, at least one of the twoLs is a nitrogen-containing axial ligand such as 5imidazole and itsderivative, pyridine and its derivative, aniline and its derivative,histidine and its derivative, and trimethylamine and its derivative, asulfur-containing axial ligand such as thiophenol and its derivative,cysteine and its derivative, and methionine and its derivative, or anoxygen-containing axial ligand such as benzoic acid and its derivative,acetic acid and its derivative, phenol and its derivative, aliphaticalcohol and its derivative, and water, and the other L is any one ofthese axial ligands or a group without a ligand.
 3. The electrodeaccording to claim 2, wherein the porphyrin compound forming the metalporphyrin complex is selected from the group consisting of5,10,15,20-tetrakis(2-thiofuryl)porphyrin,5,10,15,20-tetrakis(3-thiofuryl)porphyrin,5,10,15,20-tetrakis(2-pyrrolyl)porphyrin,5,10,15,20-tetrakis(3-pyrrolyl)porphyrin,5,10,15,20-tetrakis(2-furyl)porphyrin,5,10,15,20-tetrakis(3-furyl)porphyrin,5,10,15,20-tetrakis(2-mercaptophenyl)porphyrin,5,10,15,20-tetrakis(3-mercaptophenyl)porphyrin,5,10,15,20-tetrakis(4-mercaptophenyl)porphyrin,5,10,15,20-tetrakis(2-aminophenyl)porphyrin,5,10,15,20-tetrakis(3-aminophenyl)porphyrin,5,10,15,20-tetrakis(4-aminophenyl)porphyrin,[5,10,15-tris(2-thiofuryl)-20-mono(phenyl)]porphyrin,[5,10,15-tris(3-thiofuryl)-20-mono(phenyl)]porphyrin,[5,10-bis(2-thiofuryl)-15,20-di(phenyl)]porphyrin,[5,10-bis(3-thiofuryl)-15,20-di(phenyl)]porphyrin,[5,15-bis(2-thiofuryl)-10,20-di(phenyl)]porphyrin,[5,15-bis(3-thiofuryl)-10,20-di(phenyl)]porphyrin,[5-mono(2-thiofuryl)-10,15,20-tri(phenyl)]porphyrin, and[5-mono(3-thiofuryl)-10,15,20-tri(phenyl)]porphyrin.
 4. (Deleted) 5.(Deleted)
 6. (Deleted)
 7. (Deleted)
 8. The electrode according to claim2 or claim 3, wherein the conductive component is inserted into a smalltube prepared from an electrical insulating material, the outside ofthis small tube is covered with a material acting as a counter electrodesuch as a metal to form a counter electrode, a metal porphyrin polymermembrane is formed on the tip of the conductive component, and theelectrode has a needle-like shape.
 9. The electrode according to claim 2or claim 3, wherein the conductive component is inserted into anelectrical insulating material, the electrical insulating material isplaced in a counter electrode material, the resulting counter electrodematerial is housed in an electrical insulating material, of which theoutside is covered with a material acting as a ground, a metal porphyrinpolymer membrane is formed on the tip of the conductive component, andthe electrode has a needle-like shape.
 10. The electrode according toany one of claims 2, 3, 8, and 9, used for measuring superoxide anionradicals.
 11. A sensor for measuring the concentration of active oxygenspecies comprising an electrode for active oxygen species comprising aconductive component with a polymer membrane of a metal porphyrincomplex shown by the following formula (I) or (II) formed on thesurface, a counter electrode, and a reference electrode,

wherein M is a metal selected from the group consisting of iron,manganese, cobalt, chromium, and iridium, at least one of the four Rs isa group selected from the group consisting of a thiofuryl group,pyrrolyl group, furyl group, mercaptophenyl group, and aminophenylgroup, and the other Rs represent any one of these groups, an alkylgroup, an aryl group, or hydrogen,

wherein M and R are the same as defined above, at least one of the twoLs is a nitrogen-containing axial ligand such as imidazole and itsderivative, pyridine and its derivative, aniline and its derivative,histidine and its derivative, and trimethylamine and its derivative, asulfur-containing axial ligand such as thiophenol and its derivative,cysteine and its derivative, and methionine and its derivative, or anoxygen-containing axial ligand such as benzoic acid and its derivative,acetic acid and its derivative, phenol and its derivative, aliphaticalcohol and its derivative, and water, and the other L is any one ofthese axial ligands or a group without a ligand.
 12. The sensoraccording to claim 11, wherein the porphyrin compound forming the metalporphyrin complex is selected from the group consisting of5,10,15,20-tetrakis(2-thiofuryl)porphyrin,5,10,15,20-tetrakis(3-thiofuryl)porphyrin,5,10,15,20-tetrakis(2-pyrrolyl)porphyrin,5,10,15,20-tetrakis(3-pyrrolyl)porphyrin,5,10,15,20-tetrakis(2-furyl)porphyrin,5,10,15,20-tetrakis(3-furyl)porphyrin,5,10,15,20-tetrakis(2-mercaptophenyl)porphyrin,5,10,15,20-tetrakis(3-mercaptophenyl)porphyrin,5,10,15,20-tetrakis(4-mercaptophenyl)porphyrin,5,10,15,20-tetrakis(2-aminophenyl)porphyrin,5,10,15,20-tetrakis(3-aminophenyl)porphyrin,5,10,15,20-tetrakis(4-aminophenyl)porphyrin,[5,10,15-tris(2-thiofuryl)-20-mono(phenyl)]porphyrin,[5,10,15-tris(3-thiofuryl)-20-mono(phenyl)]porphyrin,[5,10-bis(2-thiofuryl)-15,20-di(phenyl)]porphyrin,[5,10-bis(3-thiofuryl)-15,20-di(phenyl)]porphyrin,[5,15-bis(2-thiofuryl)-10,20-di(phenyl)]porphyrin,[5,15-bis(3-thiofuryl)-10,20-di(phenyl)]porphyrin,[5-mono(2-thiofuryl)-10,15,20-tri(phenyl)]porphyrin, and[5-mono(3-thiofuryl)-10,15,20-tri(phenyl)]porphyrin.
 13. The sensoraccording to claim 11 or claim 12, wherein an electrode is used, theelectrode comprising a conductive component inserted into a small tubeprepared from an electrical insulating material, the outside of thissmall tube being covered with a material acting as a counter electrodesuch as a metal to form a counter electrode, a metal porphyrin polymermembrane being formed on the tip of the conductive component, and theelectrode having a needle-like shape.
 14. The sensor according to claim11 or claim 12, wherein an electrode is used, the electrode comprising aconductive component inserted into an electrical insulating material,the electrical insulating material being placed in a counter electrodematerial, the resulting counter electrode material being housed in anelectrical insulating material, of which the outside is coated with amaterial acting as a ground, a metal porphyrin polymer membrane beingformed on the tip of the conductive component, and the electrode havinga needle-like shape.
 15. The sensor according to anyone of claims 11-14,used for measuring superoxide anion radicals.
 16. A method for detectingactive oxygen species in a sample comprising measuring a currentproduced by oxidation-reduction reaction between a metal in a metalporphyrin polymer membrane and active oxygen species using the sensoraccording to any one of claims 11-15.
 17. The method according to claim16, wherein the active oxygen species to be detected are superoxideanion radicals.